Project Summary Central biological functions such as replication, transcription, mRNA splicing, transport, and signaling are performed by molecular machines. Standard structural techniques fail to deal with the large size of molecular complexes and provide minimal dynamic information, while fluorescence-based techniques are limited by the photo-lability and complex photophysics of conventional organic dyes. We have found that pairs of nanoparticles can be used to monitor distances via the distance- dependence of their plasmon coupling. These `plasmon rulers' reveal the time dynamics of processes such as single DNA hybridization events and the interaction of single enzymes with their DNA substrates. Suitably coated and functionalized plasmon rulers have recently enabled single-molecule studies of enzymatic DNA bending and cleavage with millisecond and nanometer resolution. The plasmon rulers did not aggregate or perturb enzyme kinetics and allowed simultaneous observation of about 5000 individual DNA substrates. The accessible distance range of plasmon rulers is 0-80nm and their photostability makes it possible to monitor single biomolecules for days. Our proposed research has three aims. First, we will optimize the optical properties of the plasmon rulers, refine nanoparticle passivation procedures, and develop improved microscopies for monitoring plasmon rulers. These technical refinements will allow researchers without specialized knowledge of plasmonics and nanoparticles to use plasmon rulers in their research. Second, we will use plasmon rulers to investigate the structural dynamics, substrate requirements, and mechanochemistry of Dicer, a central component of the RNA-induced silencing complex (RISC). RNAi is a widespread mechanism of gene regulation via post-transcriptional silencing of specific genes. RISC-assembly and -function typifies cellular processes not easily amenable to analysis by conventional methods such as FRET. Third, as a first step towards using plasmon rulers in vivo, we will establish reliable methods to deliver plasmon rulers to cells, prevent plasmon ruler aggregation once in the cytoplasm, and evaluate their possible cytotoxicity. Together, the proposed research will give biologists a new tool for monitoring single molecular machines with high temporal and spatial resolution and for nearly unlimited times. Project Narrative Public Health Significance. By developing optical probes with extreme brightness and photostability, new specific and sensitive diagnostic tools will become feasible. The single-molecule studies of RNA interference will help establish its basic mechanochemistry and may enable the development of genome-wide scans for microRNAs. Finally, the single-molecule studies of RNA interference may also facilitate the design of efficient short interfering RNA (siRNA) sequences with minimal off-target effects for therapeutic use.